Journal: Autophagy

Article Title: AP2M1 mediates autophagy-induced CLDN2 (claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability

doi: 10.1080/15548627.2021.2016233

Figure Lengend Snippet: Autophagy-mediated reduction in CLDN2/Claudin-2 levels. (A) Confluent Caco-2 cells were incubated in EBSS for starvation (ST) or treated with known autophagy inducing compounds rapamycin (RAPA, 500 nM), small-molecule enhancer 28 (SMER, 50 µM), resveratrol (RES, 100 µM), metformin (MET, 100 µM), and calcitriol (CAL, 20 µM) for 24 h, and CLDN2 levels were measured by Western blotting. ACTB/β-actin is shown as a loading control. The protein levels of CLDN2 were reduced by all the autophagy inducers. ST: starvation. The densitometry analysis of CLDN2 expression was performed using ImageJ software to indicate the relative levels of CLDN2 after autophagy-inducing treatments. The graph is representative of ≥ 3 independent experiments (*, p < 0.005 versus control). (B) The autophagy-inducing treatments increased Caco-2 trans-epithelial resistance (TER) compared to untreated control group (*, p < 0.005 versus control). (C) Autophagy inhibitor bafilomycin A1 (BAF, 20 nM) treatment for 24 h alone increased CLDN2 levels and prevented starvation-induced decrease in CLDN2 levels. (D) Autophagy inhibitor SBI-0206965 (SBI, 30 µM) treatment for 24 h prevented starvation-induced decrease in CLDN2 levels. (E) Densitometry for CLDN2 expression in panel C and D. The graph is representative of ≥ 3 independent experiments (*, p < 0.05 versus control). BAF and SBI also prevented starvation-induced increase in TER (F) (*, p < 0.005 versus control). (G) Proteasomal inhibition with MG132 (10 µM) did not prevent starvation-induced reduction in CLDN2 levels. (H) Western blot showing CLDN2 knockout in CLDN2 KO Caco-2 cells. (I) Western blot showing GFP and CLDN2 GFP, confirming CLDN2 over expression in Caco-2 cells. Starvation induced no significant difference in the levels of exogenous CLDN2 but in contrast a significant decrease in endogenous CLDN2 upon starvation was seen in both Non-Target GFP and CLDN2 overexpressing Caco-2 cells. (J) CLDN2 overexpressed Caco-2 cells (CLDN2) showed reduced TER and CLDN2 KO showed increased TER at the baseline. CLDN2 KO cells did not show starvation-induced increase in TER, whereas CLDN2-overexpressing Caco-2 cells showed mild but significant increase in TER upon starvation. a, b, c, d, and e, P < 0.005 vs. each other in two-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: CLDN2 ORF in pCMV6-AC-GFP (Origene, RG204199) and corresponding control plasmid were used to transfect Caco-2 cells using Lipofectamine 2000 (Invitrogen, 11,668,027) as per manufacturer’s instructions and the transfected cells were selected using respective antibiotic selection media to generate stable CLDN2-overexpressing Caco-2 cells.

Techniques: Incubation, Western Blot, Control, Expressing, Software, Inhibition, Knock-Out, Over Expression, Comparison

Journal: Autophagy

Article Title: AP2M1 mediates autophagy-induced CLDN2 (claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability

doi: 10.1080/15548627.2021.2016233

Figure Lengend Snippet: Autophagy-induced CLDN2 reduction is mediated via clathrin. (A) The amount of CLDN2 in membrane fraction is reduced gradually with increased starvation time period. E-cadherin-M and E-cadherin-C represents E-cadherin probes on cell membrane and cytoplasmic fractions, respectively. GAPDH is shown as a loading control for membrane fractions. The blots are representative of ≥ 3 independent experiments. (B) Amiloride (Amil, 10 µM) and Methyl-β-cyclodextrin (MβCD, 250 µM) treatment for 24 h did not alter starvation-induced reduction in CLDN2 levels. Densitometry for CLDN2 expression (*, p < 0.01 versus control). Clathrin inhibitor, chlorpromazine (CPZ, 10 µM) treatment for 24 h prevented starvation-induced reduction in CLDN2 levels (C). The densitometry representation (C) of CLDN2 expression after chlorpromazine treatment is also shown. (D) Chlorpromazine (CPZ, 10 µM) treatment for 24 h prevented starvation-induced increase in TER (*, p < 0.001 versus control) .

Article Snippet: CLDN2 ORF in pCMV6-AC-GFP (Origene, RG204199) and corresponding control plasmid were used to transfect Caco-2 cells using Lipofectamine 2000 (Invitrogen, 11,668,027) as per manufacturer’s instructions and the transfected cells were selected using respective antibiotic selection media to generate stable CLDN2-overexpressing Caco-2 cells.

Techniques: Membrane, Control, Expressing

Journal: Autophagy

Article Title: AP2M1 mediates autophagy-induced CLDN2 (claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability

doi: 10.1080/15548627.2021.2016233

Figure Lengend Snippet: Interaction of CLDN2 with clathrin and autophagy apparatus. (A) Co-immunoprecipitation studies showed an increased association of CLDN2 with AP2M1, AP2A1, clathrin, LC3 during early starvation and lysosomal marker protein LAMP2 at the later 12-h time point. The negative control includes immunoprecipitation with control IgG. (B) Quantification of CLDN2 fraction associated with various clathrin and autophagy proteins, as shown in panel A. (C) Confocal immunofluorescence examination showed that, CLDN2 (green) migrated away from the cell membrane and increased cytoplasmic colocalization with clathrin (red) after starvation (yellow). White bar: 5 µm. (D) AP2M1 immunoprecipitates showed increased presence of CLDN2, LC3 and clathrin after starvation. Representation of ≥ 3 independent experiments.

Article Snippet: CLDN2 ORF in pCMV6-AC-GFP (Origene, RG204199) and corresponding control plasmid were used to transfect Caco-2 cells using Lipofectamine 2000 (Invitrogen, 11,668,027) as per manufacturer’s instructions and the transfected cells were selected using respective antibiotic selection media to generate stable CLDN2-overexpressing Caco-2 cells.

Techniques: Immunoprecipitation, Marker, Negative Control, Control, Immunofluorescence, Membrane

Journal: Autophagy

Article Title: AP2M1 mediates autophagy-induced CLDN2 (claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability

doi: 10.1080/15548627.2021.2016233

Figure Lengend Snippet: Role of AP2M1 in regulation of CLDN2 levels. (A) AP2M1 is increasingly phosphorylated after starvation. ACTB is shown as a loading control. The blots are representative of 3 independent experiments. (B) Densitometry for phospho-AP2M1 expression in panel A (*, p < 0.01 versus control). (C) Inhibition of AP2M1 activation with Sunitinib (25 µM) treatment prevented starvation-induced reduction in CLDN2 levels. Densitometry for CLDN2 levels in Sunitinib treatment (*, p < 0.001 versus control). (D) Western blot showing efficiency of CRISPR-Cas9-mediated knockout of AAK1 in Caco-2 cells. AAK1 KO led to a significant increase in baseline CLDN2 levels and abolished starvation-induced CLDN2 reduction. (E) Densitometry for CLDN2 levels in untreated and starved non target control (NT) and AAK1 KO cells, as shown in panel D (*, p < 0.05 versus control). AAK1 knockout also significantly inhibited starvation-induced increase in TER (F) and reduction in urea flux (G) (*, p < 0.05 versus control) .

Article Snippet: CLDN2 ORF in pCMV6-AC-GFP (Origene, RG204199) and corresponding control plasmid were used to transfect Caco-2 cells using Lipofectamine 2000 (Invitrogen, 11,668,027) as per manufacturer’s instructions and the transfected cells were selected using respective antibiotic selection media to generate stable CLDN2-overexpressing Caco-2 cells.

Techniques: Control, Expressing, Inhibition, Activation Assay, Western Blot, CRISPR, Knock-Out

Journal: Autophagy

Article Title: AP2M1 mediates autophagy-induced CLDN2 (claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability

doi: 10.1080/15548627.2021.2016233

Figure Lengend Snippet: Role of AP2M1 in mice colonic TJ barrier. (A) Sunitinib administration (40 mg/kg/day for 2 days, oral gavage), causes an increase in CLDN2 levels in mouse colonocytes. Densitometry for CLDN2 levels upon sunitinib treatment (*, p < 0.005 versus control). In ex-vivo experiments in Ussing chambers, the colon of Sunitinib administered mice showed reduction in TER (*, p < 0.005 versus control) (B) and increase in urea (small molecule) flux (C) p < 0.005, compared to control mice. (D) In confocal immunofluorescence examination, sunitinib administered mice showed reduced staining for phospho-AP2M1 (red) and increased amount of CLDN2 (green) on the apical membrane of colonocytes in comparison with control mice. White bar: 20 µm. (E) Quantification of CLDN2 fluorescence from panel D (*, p < 0.01 versus control) .

Article Snippet: CLDN2 ORF in pCMV6-AC-GFP (Origene, RG204199) and corresponding control plasmid were used to transfect Caco-2 cells using Lipofectamine 2000 (Invitrogen, 11,668,027) as per manufacturer’s instructions and the transfected cells were selected using respective antibiotic selection media to generate stable CLDN2-overexpressing Caco-2 cells.

Techniques: Control, Ex Vivo, Immunofluorescence, Staining, Membrane, Comparison, Fluorescence

Journal: Autophagy

Article Title: AP2M1 mediates autophagy-induced CLDN2 (claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability

doi: 10.1080/15548627.2021.2016233

Figure Lengend Snippet: AP2M1 is required for autophagy-mediated reduction in CLDN2. (A) Western blot showing efficiency of CRISPR-Cas9 mediated deletion of AP2M1 in Caco-2 cells. AP2M1 KO led to a marked increase in baseline CLDN2 levels and abolished starvation-induced CLDN2 reduction. (B) Densitometry for CLDN2 levels in untreated and starved non target control (NT) and AP2M1 KO cells, as shown in panel A (*, p < 0.05). AP2M1 deletion also significantly inhibited starvation-induced increase in TER (C) (*, p < 0.001) and reduction in urea flux (D) (*, p < 0.001). (E) In confocal immunofluorescence examination, starvation induced increased colocalization of CLDN2 (green) and LC3 (red) when compared to control Caco-2 cells. AP2M1 KO cells showed prominent presence of CLDN2 (green) on the membrane with least colocalization with LC3 (red), even upon starvation. Nuclei: blue. White bar: 5 µm. (F) Quantification of CLDN2 and LC3 colocalization from panel E (*, p < 0.01) .

Article Snippet: CLDN2 ORF in pCMV6-AC-GFP (Origene, RG204199) and corresponding control plasmid were used to transfect Caco-2 cells using Lipofectamine 2000 (Invitrogen, 11,668,027) as per manufacturer’s instructions and the transfected cells were selected using respective antibiotic selection media to generate stable CLDN2-overexpressing Caco-2 cells.

Techniques: Western Blot, CRISPR, Control, Immunofluorescence, Membrane

Journal: Autophagy

Article Title: AP2M1 mediates autophagy-induced CLDN2 (claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability

doi: 10.1080/15548627.2021.2016233

Figure Lengend Snippet: Identification of AP2M1 anchoring region in CLDN2. (A) CLDN2 has two YXXΦ (Φ, a bulky hydrophobic residue – L/I/M/V/F) AP2M1 anchoring tyrosine motifs at amino acid 67–70 and 148–151 region. These motifs were mutated individually (67–70: CLDN2Y67A,L70A; 148–151: CLDN2Y148A,L151A) or together (Double mutant, DSDM7: CLDN2Y67A,L70A,Y148A,L151A) by site-directed mutagenesis, substituting the Y and Φ amino acids with alanine, as shown in panel A. (B) Wild type GFP-CLDN2 and the mutated GFP-CLDN2 carrying plasmids were transfected individually into HEK293 cells and co-immunoprecipitated using anti-GFP antibody. The immunoprecipitates when probed for AP2M1, showed AP2M1 being co-immunoprecipitated only with wild type CLDN2 and not with CLDN2 mutants. GFP bands are shown as loading control. IgG: Normal IgG control. Scr: scrambled control plasmid.CLDN2: wild type GFP-CLDN2. (C) In confocal immunofluorescence examination, HEK293 cells transfected with wild-type GFP-CLDN2 (CLDN2) showed strands of CLDN2 (green) and AP2 (red) colocalization. In subtracted panel, only yellow color of CLDN2-AP2 colocalization was retained. Mutations in AP2M1 anchoring motif in CLDN2 (Double mutant) altered CLDN2 localization and reduced CLDN2-AP2 colocalization, compared to wild-type CLDN2 transfected cells. Representation of 3 fields from 3 separate HEK293 monolayer samples. White bar: 10 µm. (D) Western blot showing that starvation reduces wild type GFP-CLDN2 compared to untreated control but all other mutated GFP-CLDN2 showed no significant difference compared to their corresponding untreated controls.

Article Snippet: CLDN2 ORF in pCMV6-AC-GFP (Origene, RG204199) and corresponding control plasmid were used to transfect Caco-2 cells using Lipofectamine 2000 (Invitrogen, 11,668,027) as per manufacturer’s instructions and the transfected cells were selected using respective antibiotic selection media to generate stable CLDN2-overexpressing Caco-2 cells.

Techniques: Residue, Mutagenesis, Transfection, Immunoprecipitation, Control, Plasmid Preparation, Immunofluorescence, Western Blot

Journal: Autophagy

Article Title: AP2M1 mediates autophagy-induced CLDN2 (claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability

doi: 10.1080/15548627.2021.2016233

Figure Lengend Snippet: AP2M1 at the intersection of endocytosis and autophagy. (A) Western blot shows efficacy of ATG7 knockout (ATG7 KO) in Caco-2 cells using CRISPR-Cas9. ATG7 deletion led to baseline increase in SQSTM1/p62 and CLDN2. (B) Densitometry for CLDN2 levels in panel (A) showed reduction of CLDN2 levels after starvation in non-target (NT) control cells but not in ATG7 KO cells. *, p < 0.05 versus control, NS: non-significant. (C) Co-immunoprecipitation studies using anti-LC3 antibody showed increased interaction of AP2M1 and CLDN2, with LC3 during starvation in non-target control cells. (D) Densitometry ratio of CLDN2: LC3 in panel C showed increased CLDN2-LC3 interaction after starvation in non-target (NT) control cells in comparison with ATG7 KO cells. The negative control includes immunoprecipitation with control IgG. The blots are representative of 3 independent experiments. a, b, c, and d, P < 0.01 vs. each other in two-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: CLDN2 ORF in pCMV6-AC-GFP (Origene, RG204199) and corresponding control plasmid were used to transfect Caco-2 cells using Lipofectamine 2000 (Invitrogen, 11,668,027) as per manufacturer’s instructions and the transfected cells were selected using respective antibiotic selection media to generate stable CLDN2-overexpressing Caco-2 cells.

Techniques: Western Blot, Knock-Out, CRISPR, Control, Immunoprecipitation, Comparison, Negative Control

Journal: Autophagy

Article Title: AP2M1 mediates autophagy-induced CLDN2 (claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability

doi: 10.1080/15548627.2021.2016233

Figure Lengend Snippet: Autophagy deficiency increases CLDN2 levels in-vivo. (A) Tamoxifen treatment of adult mice with Atg7 floxed and Ubc-CreERT2 alleles resulted in the loss of ATG7 protein in colonic mucosa (atg7 cKO mice) compared to ATG7 floxed (Atg7fl/fl) control mice. The Western blot also showed disruption of autophagy in atg7 cKO mice in terms of accumulation of SQSTM1/p62. Acute deletion of Atg7 also caused an increase in constitutive CLDN2 levels in the colonic epithelial cells. (B) Densitometry for CLDN2 levels in atg7 cKO mice, as shown in panel A (*, p < 0.005 versus control). The atg7 cKO mice showed reduced baseline colonic TER (C) and increased colonic urea flux (D) compared to control Atg7fl/fl mice. In acute dextran sodium sulfate (DSS) colitis model, atg7 cKO mice showed increased reduction in colonic TER and markedly increased colonic urea flux compared to Atg7fl/fl DSS mice (C and D). a, b, c, and d, P < 0.01 vs. each other in two-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: CLDN2 ORF in pCMV6-AC-GFP (Origene, RG204199) and corresponding control plasmid were used to transfect Caco-2 cells using Lipofectamine 2000 (Invitrogen, 11,668,027) as per manufacturer’s instructions and the transfected cells were selected using respective antibiotic selection media to generate stable CLDN2-overexpressing Caco-2 cells.

Techniques: In Vivo, Control, Western Blot, Disruption, Comparison

Journal: Autophagy

Article Title: AP2M1 mediates autophagy-induced CLDN2 (claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability

doi: 10.1080/15548627.2021.2016233

Figure Lengend Snippet: Role of AP2M1 in human colonic TJ barrier. Sunitinib (25 µM, 18 h) significantly decreased TER (A) and increased urea flux (B) in human colonic mucosal samples. N = 15, (*, p < 0.001 versus vehicle). (C) Sunitinib treatment reduced phospho-AP2M1 levels and increased CLDN2 levels in human colonic mucosal samples. The numbers below the individual bands indicate densitometry in terms of phospho-AP2M1:ACTB and CLDN2:ACTB ratio. (*, p < 0.01 versus vehicle) .

Article Snippet: CLDN2 ORF in pCMV6-AC-GFP (Origene, RG204199) and corresponding control plasmid were used to transfect Caco-2 cells using Lipofectamine 2000 (Invitrogen, 11,668,027) as per manufacturer’s instructions and the transfected cells were selected using respective antibiotic selection media to generate stable CLDN2-overexpressing Caco-2 cells.

Techniques:

Journal: Autophagy

Article Title: AP2M1 mediates autophagy-induced CLDN2 (claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability

doi: 10.1080/15548627.2021.2016233

Figure Lengend Snippet: Schematic model of autophagy-induced CLDN2/Claudin-2 degradation. Autophagy and AAK1 (AP2 associated kinase 1) activates AP2/Adaptor protein-2 via phosphorylation of AP2M1 (adaptor related protein complex 2 subunit mu 1). Activated AP2M1 binds to YXXΦ (AP2M1 binding site) region of CLDN2 and facilitate its CLTC/clathrin-mediated endocytosis. AP2 also contains LC3 interacting region and acts as a bridge in connecting endocytosed CLDN2 to LC3. ATG7 dependent LC3 lipidation is required for internalization of CLDN2 into autophagosomes. The CLDN2-containing autophagosome fuses with lysosome, resulting in degradation of CLDN2.

Article Snippet: CLDN2 ORF in pCMV6-AC-GFP (Origene, RG204199) and corresponding control plasmid were used to transfect Caco-2 cells using Lipofectamine 2000 (Invitrogen, 11,668,027) as per manufacturer’s instructions and the transfected cells were selected using respective antibiotic selection media to generate stable CLDN2-overexpressing Caco-2 cells.

Techniques: Phospho-proteomics, Binding Assay

Journal: Frontiers in Immunology

Article Title: IL-22 regulates endometrial regeneration by enhancing tight junctions and orchestrating extracellular matrix

doi: 10.3389/fimmu.2022.955576

Figure Lengend Snippet: Antibodies.

Article Snippet: 4 , Claudin-2, rabbit polyclonal , Novus Biologicals, CO, USA , NBP2-92140 , 1:2,000.

Techniques: Concentration Assay